Masae Tatematsu

Studies on antioxidants: Their carcinogenic and modifying effects on chemical carcinogenesis

Studies were conducted on the carcinogenic activity of butylated hydroxyanisole (BHA) in rats and hamsters. To obtain information concerning the mechanism of action of BHA on the forestomach, the following areas were examined: the effects of 12 phenolic compounds structurally related to BHA on the hamster forestomach, the effects of combinations of BHA and other antioxidants on the rat forestomach, and the metabolism of BHA in the forestomach. Also examined were the effects of several antioxidants on two-stage carcinogenesis in rats. Squamous-cell carcinomas were induced in the forestomach of rats and hamsters fed BHA. In a limited study, 1 of 13 hamsters developed a squamous-cell carcinoma. The tumorigenic action of crude BHA on the forestomach was largely due to the action of 3-tert-BHA. p-tert-Butylphenol and 2-tert-butyl-4-methylphenol induced pronounced hyperplasia and papillomas in the hamster forestomach. BHA and other antioxidants, particularly propyl gallate and ethoxyquin, showed additive effects in inducing forestomach hyperplasia and cytotoxicity. Neither BHA nor its metabolites were found in the forestomach epithelium, although small amounts of metabolites were detected in the stomach contents. Thus, a direct action on the stomach epithelium may be exerted by BHA itself or by metabolites formed on interaction of BHA with gastric juice. BHA enhanced forestomach carcinogenesis initiated in rats by N-methyl-N-nitro-N-nitrosoguanidine or N-methylnitrosourea (MNU) and enhanced urinary bladder carcinogenesis initiated by MNU or N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). In contrast, it inhibited carcinogenesis initiated in the liver by either diethylnitrosamine or N-ethyl-N-hydroxyethylnitrosamine (EHEN) and mammary carcinogenesis initiated by 7,12-dimethylbenz[a]anthracene (DMBA). BHT promoted urinary bladder carcinogenesis initiated by BBN or MNU and thyroid carcinogenesis initiated by MNU, but inhibited ear-duct carcinogenesis initiated by DMBA. Ethoxyquin promoted EHEN-initiated kidney carcinogenesis, but inhibited both DMBA-initiated mammary and EHEN-initiated liver carcinogenesis. Sodium ascorbate promoted forestomach and urinary bladder carcinogenesis, and sodium erythorbate also enhanced urinary bladder carcinogenesis. alpha-Tocopherol inhibited ear-duct carcinogenesis. No antioxidants tested had any effect on glandular stomach carcinogenesis. Thus antioxidants have independent modifying (promoting or inhibitory) effects in different organs.

Gastric and intestinal phenotypic expression of human stomach cancers as revealed by pepsinogen immunohistochemistry and mucin histochemistry

Gastric and intestinal phenotypic expression in 223 surgically obtained primary gastric cancers and their histogenetic relationship to intestinal metaplasia in the surrounding gastric mucosa were studied by mucin histochemistry and pepsinogen (Pg) immunohistochemistry. Histochemical differentiation of mucins (paradoxical concanavalin A, the galactose oxidase‐Schiff sequence and sialidase galactose oxidase Schiff) and immunohisto chemical staining of Pgs I and II, allowed differentiation of gastric cancer cells from different histological categories into gastric elements including mucous neck cells, pyloric gland cells and surface mucous cells or intestinal elements including goblet cell and intestinal absorptive cell types. Of 122 papillary and tubular adenocarcinomas, 33 (27.1%) consisted mainly of gastric type cells and 42 (34.4%) predominantly of intestinal type cells. The remainder (38.5%) consisted of mixtures of gastric‐ and intestinal‐type cells. Of 101 poorly differentiated adenocarcinomas, signet ring cell carcinomas and mucinous adenocarcinomas, 59 (58.4%) consisted mainly of gastric‐type cells and 20 (19.8%) mainly of intestinal‐type cells. Seven out of 35 papillary and tubular adenocarcinomas consisting mainly of gastric type cancer cells were surrounded by mucosa with intestinal metaplasia. Conversely, 10 out of 40 papillary and tubular adenocarcinomas consisting mainly of intestinal‐type cancer cells were observed in non metaplastic gastric mucosa. Thus no relationship as regards intestinal phenotypic expression was found between gastric cancers and surrounding gastric mucosa.

Medium-term bioassay system for detection of carcinogens and modifiers of hepatocarcinogenesis utilizing the GST-P positive liver cell focus as an endpoint marker.

We have developed a medium-term bioassay system of 8 weeks duration utilizing male Fischer 344 (F344) rats for detection of liver carcinogens and modifiers of hepatocarcinogenesis. The system consists of a single intraperitoneal injection of diethylnitrosamine (DEN, 200 mg/kg), 6-weeks-administration of test chemical beginning 2 weeks after the DEN injection, and 2/3 partial hepatectomy (PH) performed at week 3. Carcinogenic potency of test chemicals is predicted based on the results of quantitative analyses of immunohistochemically-demonstrated glutathione S-transferase placental form (GST-P) positive liver cell foci. At present, a total of 140 chemicals have been tested using this system, and the findings show a good correlation with reported carcinogenic activities in long-term tests. Furthermore, the reliability of the system has been extensively examined: the results from the medium-term bioassay were compared with those from long-term experiments using the same doses of selected chemicals; the data from presently-used 2-dimensional analysis were compared with calculated values utilizing mathematical formulae for three-dimensional analysis: conformity of phenotypic expression of enzymes in preneoplastic lesions was examined in relation to their growth activity. In conclusion, although the results with non-hepatocarcinogens were less than satisfactory, the present experimental protocol, which requires far fewer animals and shorter duration than a long-term carcinogenicity test, appears of advantage for rapid screening of the large number of environmental chemicals which may possess hazard potential for induction of liver cancer in man.

Comparative Effects of Carcinogens on the Induction of Placental Glutathione S-Transferase-positive Liver Nodules in a Short-term Assay and of Hepatocellular Carcinomas in a Long-term Assay:

The dose-dependent effects of three hepatocarcinogens were investigated by measuring the number and area of glutathione S-transferase placental form (GST-P)-positive foci and nodules appearing in the liver under short-term conditions (Experiment I) and evaluating the incidence of hepatocellular carcinoma after long-term chronic administration (Experiment II). For these purposes, three different doses of 2-acetylaminofluorene (2-AAF), 3-methyl-4-dimethylaminoazobenzene (3-Me-DAB), and DL-ethionine (ethionine) were given to male F344 rats for 6 weeks after a single injection of diethylnitrosamine (DENA) in Experiment I or for 104 weeks without initiation by DENA in Experiment II. In Experiment I, the induction of GST-P-positive foci and nodules by 2-AAF and 3-Me-DAB was clearly dose-dependent. In contrast, ethionine showed enhancing effects inducing GST-P-positive foci and nodules only in groups given the highest dose level. Similarly, in Experiment II, induction of hepatocellular carcinomas by 2-AAF and 3-Me-DAB was clearly dose-dependent, whereas liver neoplasms were only induced by the highest dose level of ethionine. These results indicate that degree of induction of GST-P positive foci and nodules in a short-term in vivo test for liver carcinogens corresponds with the incidences of hepatocellular carcinomas revealed in a long-term in vivo assay.

Placental Glutathione S-Transferase (GST-P) as a New Marker for Hepatocarcinogenesis: In Vivo Short-Term Screening for Hepatocarcinogens

Our laboratory has developed an in vivo short-term screening test for hepatocarcinogens based on quantitation of γ-glutamyl transpeptidase (γ-GT) positive foci. However, γ-GT positive hepatocytes appear in periportal areas under a variety of circumstances apparently unrelated to hepatocarcinogenesis. Glutathione S-transferase placental type (GST-P), which is hardly detectable in normal rat liver, was recently demonstrated as a new marker protein for preneoplastic liver foci. In experiment I, rats were initially given a single dose (200 mg/kg) of diethylnitrosamine intraperitoneally and 2 weeks later were treated with test compounds for 6 weeks. All rats were subjected to partial hepatectomy at week 3. The long-term development of preneoplastic lesions was followed in rats for 50 weeks. The immunohistochemical investigation of GST-P binding and the histochemical demonstration of γ-GT in serial sections revealed that almost all γ-GT foci were GST-P positive, but 5–10% of GST-P foci could not be detected by γ-GT staining. From week 8, many γ-GT foci partially lost γ-GT activity. However, no comparable disappearance of GST-P was evident in the lesions. All hepatocellular carcinomas (HC) found at week 50 consisted of GST-P positive HC cells. In contrast, 37.9% (11/29) of HC were negative for γ-GT. In experiment II (in vivo short-term screening test for hepatocarcinogens), rats were treated in the same manner as for experiment I and killed at week 8. Fifty-eight chemicals were investigated for their potential to modify GST-P positive foci development. All hepatocarcinogens and hepatopromoters clearly enhanced the induction of GST-P foci, whereas non-hepatocarcinogens and non-hepatopromoters did not. BHA and acetaminophen inhibited the development of foci. These results suggest the adoption of GST-P as a new and more accurate marker enzyme for rat liver carcinogenesis.

Induction of Adenocarcinomas in the Glandular Stomach of BALB/c Mice Treated with N‐Methyl‐N‐nitrosourea

Male 6‐week‐old BALB/c strain animals (groups 1 and 2) received 10 weekly intragastric intubations of 0.5 mg/mouse of N‐methyl‐N‐nitrosourea. At week 11 the forestomachs were resected in group 1 but not group 2. Although many animals in group 2 died due to development of squamous cell carcinomas in the forestomach, development of cancers in the glandular stomach was quite similar in both groups. Well‐differentiated adenocarcinomas in groups 1 and 2 were found at low incidence at week 20, rising to 100% at week 40, with two lesions metastasizing to the lymph nodes. Four poorly differentiated adenocarcinomas and 5 signet ring cell carcinomas were also found in 27 glandular stomach tumor‐bearing animals.

Advantages and Limitations of Stereological Estimation of Placental Glutathione S-Transferase-positive Rat Liver Cell Foci by Computerized Three-dimensional Reconstruction

The applicability to a medium‐term bioassay for liver carcinogens of mathematical formulae for the calculation of numbers of foci per volume was examined in F344 rats. Two weeks after initiation with diethylnitrosamine, animals were given test compounds for 6 weeks, partial hepatectomy being performed at week 3. At week 8, the rats were killed, the livers removed and stained immuno‐histochemically for assessment of glutathione S‐transferase P form (GST‐P)‐positive foci development. Numbers and areas of lesions were measured two‐dimensionally using a color image analyzer, and the Enzmann and Campbell formulae for estimation of number and volume per cm3 were applied to the results. In addition, three‐dimensional reconstruction of individual foci was performed using up to 150 GST‐P stained foci, with the aid of a computerized graphic system. Both two‐ and three‐dimensionally expressed quantitative results were found to adequately demonstrate the modifying potential of test chemicals on hepatocarcinogenesis. The three‐dimensional approach was only more accurate if most of the foci were small and the liver was enlarged by compound treatment. Stereological reconstruction revealed that the shape of GST‐P‐positive foci, especially if relatively large, is not always spherical but that many demonstrate irregular branching forms, so that the assumptions behind Stereological estimation are not met. The results therefore show that care must be taken in applying mathematical formulae for the calculation of three‐dimensional data.

Differentiation of pepsinogen-producing cells in the fundic and pyloric mucosa of developing rats

Abstract In the rat fundic mucosa, increase in the peptic activity of pepsinogen, changes in the molecular species of pepsinogen separated by electrophoresis and changes in the morphology of the chief cells indicated that maturation of the chief cells began around 15 days after birth, and was complete 25–30 days after birth. In the rat pyloric mucosa, no changes in the peptic activity or molecular species of pepsinogen or in the morphology of pyloric gland cells occurred after birth. These results suggest that maturation of pyloric gland cells is complete before birth.

Dose-dependent effects of butylated hydroxyanisole, butylated hydroxytoluene and ethoxyquin in induction of foci of rat liver cells containing the placental form of glutathione S-transferase

The dose-dependence effects of 3 antioxidants, butylated hydroxyanisole (BHA: 2.0%, 1.0% and 0.5%), butylated hydroxytoluene (BHT: 1.0%, 0.5% and 0.25%) and ethoxyquin (EQ: 0.5%, 0.25% and 0.125%) combined with partial hepatectomy on the development of preneoplastic lesions in the liver of diethylnitrosamine (DEN, 200 mg/kg body wt)-treated rats were investigated. Feeding of the antioxidants commenced 2 weeks after the single dose of DEN used to initiate the lesions. gamma-Glutamyl transpeptidase (gamma-GT) and the placental form of glutathione S-transferase (GST-P) were used for quantitation of altered focal populations. Results with both markers demonstrated a dose-dependent decrease of foci in BHA-treated rats relative to those in control rats. Morphometric analysis of gamma-GT-positive lesions also revealed decrease in both the number and area of foci in BHT- and EQ-treated groups. The discrepancy between results of quantitation of gamma-GT- and GST-P-positive foci was attributable to the induction of a background, periportal zone staining for gamma-GT, which made differentiation of smaller foci difficult. Comparison of results with the 2 markers suggested that GST-P is the more accurate marker for quantitative studies on enzyme altered foci in rat liver.

Cellular differentiation and histogenesis of rat glandular stomach cancers.

The gastric and intestinal phenotypic expressions of tumor cells in 18 adenomatous hyperplasias, 33 well‐differentiated adenocarcinomas, and 16 undifferentiated adenocarcinomas (4 poorly differentiated adenocarcinomas, 10 signet‐ring cell carcinomas and 2 mucinous adenocarcinomas) induced by N‐methyl‐N’‐nitro‐N‐nitrosoguanidine or 4‐nitroquinoline‐I‐oxide in the rat glandular stomach were studied by histochemical stainings for mucin and immunohistochemical staining for pepsinogen isozyme 1 (Pg 1). By histochemical staining for mucin [by the paradoxical concanavalin A method, the modified method with labeled peanut lectin, the galactose oxidase‐Schiff (GOS) reaction, and the sialidase‐GOS reaction] and immunohistochemical staining of Pg 1, gastric cancer cells of each histological group could be clearly classified into a gastric type, including mucous neck cell pyloric gland cell, and surface mucous cell subtypes, and an intestinal type, including goblet‐cell, and intestinal absorptive cell subtypes. All tumors examined in this work consisted mainly of gastric‐type cells but intestinal‐type tumor cells were occasionally found among the gastric‐type tumor cells. The incidences of intestinal‐type cells in adenomatous hyperplasias (11.1%) and small well‐differentiated adenocarcinomas (28.6%) were significantly less (P<0.05) than that in large well‐differentiated adenocarcinomas (68.4%). The incidence of intestinal‐type cells in small undifferentiated adenocarcinomas (25.0%) was also less than that in large ones (58.3%). The present results suggest the occurrence of change of phenotypic expression of tumor cells from the gastric type to the intestinal type during growth of tumors.